Preparation of the formaurindicarboxylic acid base and its derivations and use

ABSTRACT

A pharmaceutical composition comprising at least 0.1 μmol of formaurindicarboxylic acid or its derivatives in 1 kg of pharmaceutically acceptable carrier. The pharmaceutical composition of claim  1  wherein the composition is in the form of solution prepared using aqueous alcali or water.

FIELD OF INVENTION

The present invention relates to the preparation for prevention andtherapy of coccidial infections, against which formaurindicarboxylicacid and its derivates are used as highly effective anticoccidial agent.

BACKGROUND OF THE INVENTION

Coccidia parasites infect a wide range of animal species, especiallyfarm animals where they cause significant economic losses due tomortality, morbidity, lower performance and extra costs associated withtreatment and prevention. The most common coccidia belong to the genusEimeria, which infect birds and mammals. Other common coccidian generafound in farm animals are Cryptosporidium and Isospora.

Cryptosporidium parvum typically infects the small intestine of neonatesof ruminants, and is also human pathogen. In the human population, C.parvum and the genetically closely related species C. hominis are mostlydiagnosed in individuals with a compromised immune system, mostly peoplewith AIDS, where they cause profuse, long-lasting watery diarrhea. Inimmunocompetent people Cryptosporidium causes enteritis and diarrheawhich is self-limiting. Nevertheless, severe diarrhea and dehydrationmay cause serious complications in children and in older persons.

Today, two different approaches for the control of coccidioses areused: 1) vaccination, and 2) administration of coccidiostats andanticoccidials. Vaccination against some Eimeria species is commerciallyavailable for use in poultry. Other animals must be treated byanticoccidials covering now all known coccidian species. A problemaccompanying the use of these drugs, which were mostly synthesized in1950s-70s, is the risk of resistancy and the need to rotate treatmentprograms. Also, residuality of these drugs represents a risk if drugsenter the food chain.

Treatment of cryptosporidial infections is problematic due to the lackof reliable drugs, as well as by the limitations associated withimmunotherapy (Harp et Goff, 1995; Jenkins, 2004). People may be treatedwith paromomycin, and by azithromycin, nitazoxanide or lehrazuril, drugswith a limited efficacy against cryptosporidiosis (Blanshard et al.,1997; Giacometti et al., 1998). In animals, the only registered drug ishalofuginone-lactate.

Because of these problems, it is evident that there is a need to findnew highly effective anticryptosporidial drugs which should not be toxicto the host and should not leave any residual.

BACKGROUND PUBLICATIONS INCLUDE

Blanshard C, Shanson D C, Gazzard B G: Pilot studies of azithromycin,letrazuril and paromomycin in the treatment of cryptosporidiosis. Int JSTD AIDS 1997; 8(2):124-9

Giacometti A, Burzacchini F, Cirioni O, Barchiesi F, Dini M, Scalise G:Efficacy of treatment with paromomycin, azithromycin, and nitazoxanidein a patient with disseminated cryptosporidiosis. Eur J Clin MicrobiolInfect Dis, 1998; 18:885-889.

Harp J A, Goff J P: Protection of calves with a vaccine againstCryptosporidium parvum. J Parasitol. 1995; 81(1):54-7.

Jenkins M C: Present and future control of cryptosporidiosis in humansand animals. Expert Rev Vaccines. 2004; 3(6):669-71

Liang F, Huang Z, Lee S Y, Liang J, Ivanov M I, Alonso A, Bliska J B,Lawrence D S, Mustelin T, Zhang Z Y: Aurintricarboxylic acid blocks invitro and in vivo activity of YopH, an essential virulent factor ofYersinia pestis, the agent of plague. J Biol Chem. 2003;278(43):41734-41.

Okada N, Koizumi S: A neuroprotective compound, aurin tricarboxylicacid, stimulates the tyrosine phosphorylation cascade in PC12 cells. JBiol Chem. 1995; 270(27):16464-9.

Owens M R, Holme S.: Aurin tricarboxylic acid inhibits adhesion ofplatelets to subendothelium. Thromb Res. 1996; 81(2):177-85.

Tzipori S: Cryptosporidiosis: laboratory investigations andchemotherapy. Adv Parasitol. 1998; 40:187-221

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

The above mentioned drawbacks of existing treatments are eliminatedusing a preventive and therapeutical treatment against coccidioses bythe invention, wherein 1 kg of suitable carrier contains at least 0.1μmol of formaurindicarboxylic acid and its derivates represented by thefollowing formula:

-   -   in which R1-R3 mean

R1 R2 R3 Formaurindicarboxylic acid H COOH COOH Formaurindicarboxylates(salts of H COO⁻ COO⁻ formaurindicarboxylic acid) Aurintricarboxylicacid

COOH COOH Aurintricarboxylates (salts of aurintricarboxylic acid)

COO⁻ COO⁻ Aluminon

COO⁻NH₄ ⁺ COO⁻NH₄ ⁺

The carrier can be any suitable material, including aqueous alcalicsolution or water.

The above mentioned derivative compounds are known and the mostimportant of them, aurintricarboxylic acid (ATA) has been described forits strong biological effects on organisms. ATA is a potent inhibitor ofendonuclease activity; namely DNAase I, RNAase A, S1 nuclease,exonuclease III or restriction endonucleases Sal I, Bam HI, Pst I andSma I (Hallick et al. 1977). Other biological properties of ATA arebased on its ability to stimulate tyrosin phosphorylation (Okada andKoizumi, 1995). ATA also inhibits von Willebrand factor binding toplatelets and thus prevents blood coagulation (Owens and Holme, 1996).Antimicrobial properties of ATA have been also described as thiscompound suppresses pathogenicity of Yersinia (Liang et al., 2003).

Interestingly, the use of aurintricarboxylic acid for antiprotozoaltreatment has not been described to date. In the literature, thesecompounds are described as aurintricarboxylic acid (CAS#4431-00-9),triammonium salt of aurintricarboxylic acid, aluminon (CAS#569-58-4),and as formaurindicarboxylic acid (CAS#621051-06-3).

Because of the mechanism of action of these compounds, there is reasonto believe that may also be used against other protozoal infections, ifadministered in an appropriate formulation.

EXAMPLE

In vivo model designed by Tzipori (1998) was used in followingmodification: Three-day-old, SPF C57B1/6 baby mice (Velaz, CzechRepublic) were randomly assigned to groups of 8-9 mice. The baby micewere caged with their mother in sterile microisolators equipped withfilter tops under standard conditions. 4-day-old mice were orallyinfected with 100 000 oocysts and the treatment was initiated at thesame time; the daily dose of aurintricarboxylic acid 50 and 100mmol/kg/day was divided into 2 equal portions and orally administered in12-hr intervals. A control group received phosphate buffer saline, andparomomycin (100 mg/kg/day) was used as positive control. ATA wasprepared fresh before each use by dissolving the drug in aqueoussolution of sodium hydroxide or potassium hydroxide at a molar ratio of1:3 (ATA:NaOH), or in dimethylsulfoxide. The treatment was continued for8 days and it was tolerated without any visible signs of toxicity.Intensity of infection was expressed as the number of oocysts inhomogenized intestinal tract in the 9th day after infection. Theexperiment was repeated between 2006 and 2007 and always gave verysimilar results. FIG. 1 shows results of the last experiment where ATAwas dissolved in sodium hydroxide.

There is reason to believe that ATA is also effective against othercoccidians (including Eimeria, Isospora).

Structure of formaurindicarboxylic acid and its derivates of thestructure:

-   -   In which R1-R3 mean

R1 R2 R3 Formaurindicarboxylic acid H COOH COOH Formaurindicarboxylates(salts of H COO⁻ COO⁻ formaurindicarboxylic acid) Aurintricarboxylicacid

COOH COOH Aurintricarboxylates (salts of aurintricarboxylic acid)

COO⁻ COO⁻ Aluminon

COO⁻NH₄ ⁺ COO⁻NH₄ ⁺

The inihibition aurintricarboxylic acid on experimental Cryptosporidiumparvum—infection in suckling C57B16 mice is graphed in FIG. 1.

1. A pharmaceutical composition suitable for treatment of a bird ormammal subject to coccidial infection by cryptosporidium speciescomprising a pharmaceutically effective amount of at least one compoundselected from the group consisting of formaurindicarboxylic acid,aurintricarboxylic acid, salts of formaurindicarboxylic acid andaurintricarboxylic acid, and aluminon, effective againstcryptosporidiosis, in a pharmaceutically acceptable carrier.
 2. Thepharmaceutical composition of claim 1 wherein the composition is in theform of solution prepared using aqueous alkali or water.
 3. Thepharmaceutical composition of claim 1, wherein the pharmaceuticallyeffective amount comprises at least 0.1 μmol of the at least onecompound in 1 kg of the pharmaceutically acceptable carrier.